Multiple Episodes of Convergence in Genes of the Dim Light Vision Pathway in Bats Yong-Yi Shen1,2, Burton K. Lim3, He-Qun Liu1,4, Jie Liu1,4, David M. Irwin5,6, Ya-Ping Zhang1,2* 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming, China, 2 Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, China, 3 Department of Natural History, Royal Ontario Museum, Toronto, Canada, 4 Graduate School of the Chinese Academy of Sciences, Beijing, China, 5 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada, 6 Banting and Best Diabetes Centre, University of Toronto, Toronto, Canada Abstract The molecular basis of the evolution of phenotypic characters is very complex and is poorly understood with few examples documenting the roles of multiple genes. Considering that a single gene cannot fully explain the convergence of phenotypic characters, we choose to study the convergent evolution of rod vision in two divergent bats from a network perspective. The Old World fruit bats (Pteropodidae) are non-echolocating and have binocular vision, whereas the sheath- tailed bats (Emballonuridae) are echolocating and have monocular vision; however, they both have relatively large eyes and rely more on rod vision to find food and navigate in the night. We found that the genes CRX, which plays an essential role in the differentiation of photoreceptor cells, SAG, which is involved in the desensitization of the photoactivated transduction cascade, and the photoreceptor gene RH, which is directly responsible for the perception of dim light, have undergone parallel sequence evolution in two divergent lineages of bats with larger eyes (Pteropodidae and Emballonuroidea). The multiple convergent events in the network of genes essential for rod vision is a rare phenomenon that illustrates the importance of investigating pathways and networks in the evolution of the molecular basis of phenotypic convergence. Citation: Shen Y-Y, Lim BK, Liu H-Q, Liu J, Irwin DM, et al. (2012) Multiple Episodes of Convergence in Genes of the Dim Light Vision Pathway in Bats. PLoS ONE 7(4): e34564. doi:10.1371/journal.pone.0034564 Editor: Eric James Warrant, Lund University, Sweden Received September 19, 2011; Accepted March 2, 2012; Published April 11, 2012 Copyright: ß 2012 Shen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Basic Research Program of China (973 Program, 2007CB411600), National Natural Science Foundation of China (30621092 and 31172080), Bureau of Science and Technology of Yunnan Province, and research grants from the ROM Governors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction explain in part why both Emballonuridae and OW fruit bats both have well-developed visual systems [17]. The independent Independent convergent evolution of phenotypic characters in development of large eyes in Pteropodidae and Emballonuridae response to similar selective pressures is not rare, however the may reflect a functional convergence on the use of rod vision. Rod molecular basis of these phenomena are poorly known [1,2]. In vision involves several processes, including light sensing, signal previous studies, a single gene is often used to explain the transduction, and interpretation in the brain cortex, among others, phenotypic convergence of echolocation in mammals and dim- and thus many genes are involved [18,19]. A previous study light vision in bats [3,4,5]. The genetic makeup of these indicated that convergent evolution in rhodopsin (RH1), a rod phenotypic characters is very complex and undoubtedly many vision gene that encodes the pigment directly responsible for the other genes are involved in both echolocation and vision. For perception of dim light [20], had occurred in Pteropodidae and example, dim-light vision requires a series of genes, not only the Emballonuridae [5]. Rod vision requires many other genes, such visual pigment genes, but also genes that are involved in the as CRX, which encodes the cone-rod homeobox protein that is a desensitization of the photoactivated pigments, photoreceptor photoreceptor-specific transcription factor essential for the differ- development, and visual signal transduction. Multiple genes are entiation of photoreceptor cells [21]. CRX regulates the expression essential for functional rod vision. of many rod vision-specific genes [22], and mutations in this gene Bats are adapted to a nocturnal niche; however, their reliance cause autosomal dominant cone-rod dystrophy [23], autosomal on vision varies among species. Old World fruit bats (family dominant retinitis pigmentosa [24] and Leber’s congenital Pteropodidae), for example, do not have laryngeal echolocation amaurosis [25,26]. Another gene involved in rod vision is SAG, [6], and instead navigate largely by sight with larger eyes and which encodes S-arrestin protein, a major soluble photoreceptor binocular vision [7,8,9,10]. Other types of bats have laryngeal protein that is involved in the desensitization of the photoactivated echolocation, and in general have smaller eyes with monocular transduction cascade. Mutations in SAG are associated with night vision [11,12,13]. An exception to this are the sheath-tailed bats blindness [27,28]. While RH1 is essential for perception in dim (Emballonuridae) who have relatively large eyes and appear to light, CRX and SAG are also of critical importance for the function have a greater reliance on visual sight compared to most bats of photoreceptor cells and the animal’s ability to adapt to dim [14,15]. The Emballonuridae with their relatively large superior light. colliculi resemble OW fruit bats in this respect [16], which may PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34564 Convergent Evolution in Rod Vision Genes In this study, we amplified and sequenced CRX and SAG genes P = 0.06, see Table S1), with the sites 133P (pp = 0.888) and 242V from 38 individuals representing 29 species across the five major (pp = 0.976) on these lineages being the positively selected sites. groups of bats (Emballonuroidea, Noctilionoidea, Pteropodidae, Rhinolophoidea, and Vespertilionoidea). Similar to previous The parallel sequence evolution of SAG findings with RH1 [5], we found evidence supporting convergent SAG genes were successfully amplified from the 25 individuals evolution in both CRX and SAG, providing a rare example of representing 18 species of bats. The amplified SAG sequence multiple events of convergent evolution occurring in parallel in corresponds to bases 181 to 951 of the 1218 base coding sequence interrelated genes, suggesting that multiple changes are involved in of the human gene. No insertion/deletion mutations or changes the network of genes necessary for rod vision to generate the that result in stop codons were found in any of the sequences, complex molecular and phenotypic convergences. suggesting that all bats have a functional SAG gene. The aligned SAG nucleotide sequences were 771 bp in length, including a 3 bp Results insertion, of which 210 were variable and generated 55 sites with amino acid variations (Figure S5). Phylogenetic trees generated The parallel sequence evolution of CRX genes from the nucleotide (771 bp) and amino acid (257 sites) sequences CRX genes were amplified from 38 individuals representing 29 of the aligned SAG gene by multiple methods resulted in trees that species of bats. The aligned nucleotide sequence was 861 base were congruent with the best-supported phylogeny of bats pairs (bp) in length, of which 287 were variable and generated 83 generated from other data sources [29,30] (Figure S6). sites with amino acid variation (Figure S1). The amplified CRX Ancestral SAG sequences for the internal nodes of the species sequence corresponds to bases 25 to 879 of the 897 base coding tree were reconstructed and amino acid substitutions were inferred sequence of the human gene. No insertion/deletion mutations or onto each lineage. Pteropodidae and Emballonuroidea were both change that resulted in a stop codon were found in any of the found to share an I51M amino acid replacement (marked in bold sequences, suggesting that all of the bats have a functional CRX black, Figure 2) at a site that is conserved among examined gene. mammals (Figure S5). The probability that this parallel evolu- Phylogenetic analyses of the aligned CRX nucleotide sequences tionary change occurred by random on these two branches was (861 bp) with Bayesian, Maximum Likelihood and Neighbor- significantly rejected (P = 0.011) by a statistical test [31]. joining methods resulted in consistent trees that were congruent Additional parallel replacements were observed between different with the best-supported species tree [29,30] (Figure S2). While bat lineages (e.g., T201S, K99R, T95A, and E96D), however they Pteropodidae and Emballonuridea are two divergent lineages of occurred on other branches that had no obvious shared bats, the phylogenetic tree generated from amino acid sequence morphological or ecological similarity and as these were sites